New methods in diagnosis and therapy of viral diseases, chronic pathogen infections generally and cancer emerge when the diverse roles in host defense played by natural dsRNA are uncovered. For example, development of AIDS (a retroviral infection/cancer) is associated with progressively severe dysfunction in biological processes which I have found to be catalyzed by dsRNA including not only interferon biosynthesis, but also bioactive 2-5A production, RNase L activity and various cell-mediated immune functions. I show that specific reduction in bioactive dsRNA, or enzymes which depend on dsRNA, within specific cells contributes to disease progression.
A deficiency state in pathways involving bioactive dsRNA also lies at the core of various cellular lesions in host defense systems leading to death. Earlier, I detected an inhibitor of RNase L in lymphocytes of HIV-infected ARC and AIDS patients which is just one result (from many) of inadequate intracellular dsRNA levels. Therapy with appropriately configured synthetic dsRNA eliminates this inhibitor(s), which can explain part of the clinical responses I observed with respect to reduction in virus concentration and restoration of immune function in patients with a variety of chronic viremias which at present continue to largely defy definitive medical intervention. Accordingly, I have now developed an integrative invention which can explain, quantitate, and correct a variety of dsRNA deficiency states in varied disease settings. I have also discovered that certain dsRNAs, e.g., mismatched dsRNA, can actually substitute for a naturally occuring dsRNA which help maintain functions essential for normal cell processes. The absence of such dsRNA regulation, if not corrected with a suitable exogenous dsRNA, can cause various abnormal cellular processes to develop. Abnormal cellular processes then lead to chronic viral or other intracellular pathogen infections, reduced immune cell functions and, ultimately, chronic morbidity and possibly death itself.
Clinical Strategies. Viral infections can often follow partial disturbance of the immune system through agents as diverse as stress and viruses which directly attack immune function. For example, AIDS (Acquired Immune Deficiency Syndrome) follows the progressive deterioration of the immune system caused by infection of T lymphocytes by human immunodeficiency virus, HIV (Coffin et al., Science vol. 232, p. 697, 1986).
For completeness, it should be noted that different designators for the HIV virus exist; LAV is the designator for the AIDS virus isolated at the Pasteur Institute, Paris, France and HTLV-III is the designator for the AIDS virus isolated at the National Institute of Health, Bethesda, Md., U.S.A. Currently, HIV is used as a generic term for the virus. Frequently in this text, the HIV virus will be referred to generically or designated HIV or HTLV-III or LAV without intending to differentiate between them. Furthermore, the term HIV in the specification and claims includes all other viruses which may be associated with producing AIDS, whether yet isolated or not.
HIV infection is a progressive disease although the rate of transition from one phase to another is variable. Asymptomatic individuals infected by HIV can develop a condition (LAS or pre-ARC) characterized by lymphadenopathy. Patients progress to AIDS-related complex or ARC by exhibiting the T4 cell deficit and later show reduced ability of lymphocytes to undergo antigen-stimulated proliferation and IFN-gamma production. These patients lose various cell mediated immune functions such as delayed cutaneous hypersensitivity, eventually becoming completely anergic. They exhibit evidence of breakdown in host defense mechanisms including Herpes Zoster infections, oral candidiasis (thrush), and symptoms such as prolonged fevers, night sweats diarrhea and weight loss. Finally, these patients experience certain severe opportunistic infections (e.g., Pneumocystis carinii pneumonia) and are then defined as having full-blown AIDS, with an approximately 50% mortality within 12 months. The HIV virus infection is chosen as an illustrative case simply because the level of dsRNA deficiency is so pronounced. Many other diseases, including indolent viral infections, will be associated with similar deficiencies; thus, my invention has unexpected wide applicability.
Importantly, the typical progression outlined above does not describe the remarkable disparity in clinical symptomatology among HIV-infected patients or many other patients with chronic disease such as Epstein Barr or EB infection, hepatitis virus infection, cytomegalovirus, herpes infections, etc. Components of the immune system decay with vastly different rates in various patients and the lack of bioactive dsRNA is a cause or contributing factor in this process. HIV infection may develop in a variety of different types of cells (e.g., blood cells, glial cells). Some patients develop neurological dysfunction as their first symptom. Thus, various individuals may have vastly different therapeutic requirements even though all will probably require HIV eradication from T4 cells.
Two general pathological features need to be addressed in many different viral infections including those of ARC patients: a collection of immune deficits and ongoing viral infection (reviewed in Fauci, Proc. Nat'l. Acad. Sci. USA, vol. 83, p. 9278, 1986). Therapeutic intervention which attacks only the immune deficits but fails to control virus replication appears to be counterproductive especially when such treatment activates T4 cells. For example, T-helper cells with Tac receptors are responsive to the lymphokine IL-2. In AIDS victims, IL-2 causes expansion of HIV-sensitive cells and apparent worsening of the disease.
Attacking viral infections directly, e.g., the HIV infection, has met with some success especially evidenced by the viral reverse transriptase inhibitor designated azidothymidine (AZT) which can reduce HIV "load" in some patients and prolong life. Such therapy is not without undesirable results. In a minority of patients, AZT also causes temporary recovery of T-cell meditated immunity as measured by a transient rise in T4 cells and by appearance of delayed type hypersensitivity (Fischl et al. New Eng. J. Med., July, 1987) However, reverse transcriptase inhibitors are toxic at doses needed to produce significant antiviral effects and are thus poorly tolerated over time. Antiviral agents may need to be administered for the life of the patient.
IFN is both ineffective as a general antiviral compound and as an anti-AIDS drug, probably because it is not an antidote for the dsRNA-relted defects which I have now uncovered. Certainly, ARC/AIDS victims have various "defects" in IFN pathways, such as the production of a defective, acid-labile IFN and inability of T4 cells to produce appropriate amounts of IFN gamma which are well described in the medical literature. Earlier, I concluded that these various IFN-related defects in ARC and AIDS patients may prove to be related in whole or part to a lack of normal RNase L (a terminal pathway mediator) activity in lymphocytes which I described in my copending application Ser. No. 021,372 filed Mar. 3, 1987, which is hereby incorporated by reference. Although dsRNA-dependent 2-5A synthetase is elevated in blood lymphocytes of individuals with ARC/AIDS as well as other chronic viremias, I have found that the same blood cells show diminished levels of authentic 2-5A and low RNase L (see below). I correlated the low RNase L activity with low quantities of M.sub.r 80,000 protein capable of binding a photoaffinity labeled analogue of 2-5A. My findings are consistent with a dsRNA pathway(s) which has (have) been shut down and the relative absence and/or inhibitor of RNase L.
Vaccination against HIV and other viruses, and the use of passive anti-HIV antibodies, are also under consideration for viral prevention and treatment, respectively, but may have significant limitations: for example, HIV (as well as influenza virus in particular) readily can infect cells covertly. I also described a role for dsRNA to enhance antibody formation against viruses in general and retroviruses in particular in the above-mentioned patent application.
I describe herein a new phenomenon that argues for double-stranded RNAs (dsRNA) to constitute the first group of molecules which are effective against both the viral and immune lesions of various subacute/chronic vital infections such as HIV and others. These defects can be corrected with laser-like precision without adversely affecting other bodily functions which is a common limitation of presently available therapies for chronic viral infections. I conclude that patients with unresolved and indolent viral infections often have specific defects in vital intracellular dsRNA-dependent pathways which can be reversed or ameliorated at least in part by an exogenously supplied dsRNA (this is explained graphically in the flow chart of FIG. 1), and that monitoring these pathways, before and during treatment, provides a novel efficacious manner to gauge the degree of dsRNA replacement therapy required an individual patient or specific disease basis.
Critical enzymes associated with host defense require dsRNAs for expressing bioactivity. These enzymes can be activated by exogenous dsRNA if the level of natural intracellular dsRNA is too low. Part of the pleiotropic action of dsRNA derives from its ability to induce the synthesis of the entire range of IFNs--alpha, beta and gamma--which operate through a group of intracellular mediators including a dsRNA-dependent protein kinase, dsRNA-dependent 2-5A synthetase and 2-5A-dependent RNase L. These dsRNA-dependent enzymes may carry out many biological activities attributable to IFN as reviewed by Lengyel (Ann. Rev. Biochem., vol. 51, p. 251, 1882). For example, production of an antisense RNA which blocks expression of 2-5A-synthetase causes profound sensitivity of animal cells to infection by different viruses (Bendetti et al. Prac. Natl. Acad. Sci USA, vol. 84, p. 658, 1987). The dsRNA-activated protein kinase phosphorylates e1F2 which inhibits protein syntheses; it also has proteolytic activity to degrade viral proteins. DsRNA-activated 2-5A synthetase synthesizes 2-5A which activate RNase L, a pathway which I suggest plays a crucial role both in the inhibition of various animal viruses and in cell growth control (escape from which may lead to tumor formation). 2'-5' oligoadenylic acids are unusual in that the normal 3',5'phosphodiester linkage characteristics of most dsRNA has been replaced by a novel phosphodiester bond with acquisition of new biological properties.
Antiviral Activity of dsRNA. dsRNA is a well-known inducer of an antiviral state (Marcus et al, Nature, vol. 66, p. 815, 1977). Similarly, poly(A):poly(U) generated an antiviral state without inducing IFN. But, prior to my current invention, it was not known that natural dsRNA regulators exist and, therefore, that actual dsRNA deficiency states are common in humans, particularly associated with pathogenic episodes (viral, fungal, protozoan, bacterial invasions, etc., especially those which have intracellular (human) presence as a valuable or necessary components of their life cycle).
Antiproliferative Activity of dsRNA. Earlier, I found (J. IFN. Res., vol. 6, p. 373, 1986) that 42% of more than 100 fresh human tumor specimens, when studied in soft agar, showed a 50% or greater reduction in tumor cell colony formation after only one exposure to dsRNA. These cells are dsRNA deficient. I also described independent IFN and dsRNA sensitivity in various human tumor cell lines and found that human tumors propagated in nude mice were sensitive to dsRNA; dsRNA therapy was curative of some tumors (e.g., renal) and the animals lived to a normal life expectancy (see published European patent application 0,113,162). Herein, I report a strong correlation between clinical responses and dsRNA-dependent enzyme activation as opposed to detectable IFN or IL-2 induction secondary to dsRNA administration.
Immune Enhancement Activity of dsRNA. Earlier I reported (J. Immunol., vol. 124, p. 1852, 1980) that dsRNA increased human natural killer NK cytlytic activity against human leukemia cells. The dsRNA structural requirements for NK augmentation paralleled those needed for 2-5A synthetase activation and for IFN induction.
Gene Control Mediated by dsRNA. Sullo et al (Cell, vol. 43, p. 793, 1985) demonstrated that dsRNA induces competence genes in resting fibroblasts including the oncogenes designated fos and myc. As used herein, "competence genes" are those genes in a cell whose actions are required in order to modulate vital actions such as multiplication, growth, etc. The IFN-beta gene induction by dsRNA may utilize a regulatory protein which is removed upon exposure of cells to dsRNA (Zinn et al. Cell, vol. 45, p. 611, 1986). I have determined that, to biological activity, exogenously supplied dsRNAs mimic a natural, nonviral, intracellular dsRNA such as heterogeneous nuclear RNA or mRNA complexed with poly(U). For example, I recently discovered an IFN-inducible dsRNA in Hela cell nuclei capable of activating 2-5A synthetase in vitro. The advent of cellular immunity far back in the evolutionary cycle in animals apparently set the stage for the evolution of IFN/dsRNA molecules toward implementing cellular differentiation, particularly differentiation promoted by IFN, I was able to achieve the more general insight that dsRNA can implement differentiation promoted by other regulatory proteins which set the stage for my definition of dsRNA deficiency states.